Characterization of Leachianone G 2″ -Dimethylallyltransferase, a Novel Prenyl Side-Chain Elongation Enzyme for the Formation of the Lavandulyl Group of Sophoraflavanone G in Sophora flavescens Ait. Cell Suspension Cultures

Abstract

Leachianone G (LG) 2ЈЈ-dimethylallyltransferase, a novel prenyl side-chain elongation enzyme, was identified in Sophora flavescens Ait. cultured cells. The enzyme transfers a dimethylallyl group to the 2ЈЈ position of another dimethylallyl group attached at position 8 of LG to form sophoraflavanone G, a branched monoterpenoid-conjugated flavanone characteristic to this plant. This membrane-bound dimethylallyltransferase required Mg 2ϩ (optimum concentration was 10 mm) for the reaction and had an optimum pH of 8.8. It utilized dimethylallyl diphosphate as the sole prenyl donor, and the 2Ј-hydroxy function in LG was indispensable to the activity. The apparent K m values for dimethylallyl diphosphate and LG were 59 and 2.3 m, respectively. Subcellular localization of three enzymes that participated in the formation of the lavandulyl group was also investigated by sucrose density gradient centrifugation. Two prenyltransferases, naringenin 8-dimethylallyltransferase and LG 2ЈЈ-dimethylallyltransferase, were localized in the plastids, whereas 8-dimethylallylnaringenin 2Ј-hydroxylase, which catalyzes the crucial step in the lavandulyl-group formation, was associated with the endoplasmic reticulum. These results suggest the close cooperation between the plastids and the endoplasmic reticulum in the formation of lavandulyl groups.More than 30,000 isoprenoid compounds-the most chemically diverse family of metabolites-are found in nature (Eisenreich et al., 1998). The vast majority of these compounds have "regular" 1Ј-4 (head-to-tail) linkages between isoprenoid units formed by isoprenyl diphosphate synthases, which are types of prenyltransferase. These enzymes catalyze the prenyl diphosphate elongation reaction that results in the consecutive 1Ј-4 condensations of isopentenyl diphosphate (IPP), the five-carbon building unit, with allylic isoprenyl diphosphates such as dimethylallyl diphosphate (DMAPP) and geranyl diphosphate (GPP; Ogura and Koyama, 1998; Liang et al., 2002). "Irregular" (non-head-to-tail) isoprenoids are also found, of which the most prominent examples are the 1Ј-1 (tail-to-tail) condensed isoprenoids such as squalene and phytoene, which are important precursors of sterols and carotenoids, respectively (Poulter, 1990). The biosyntheses of these tail-to-tail condensed terpenoids have been well characterized, but little is known about other nonhead-to-tail condensed terpenoids except for chrysanthemyl diphosphate synthase, which catalyzes the condensation of two molecules of DMAPP to produce chrysanthemyl diphosphate, a branched monoterpene with a c1Ј-2-3 linkage between two isoprenoid units (Poulter, 1990;Rivera et al., 2001). A lavandulyl group, another example of the branched monoterpenoid unit, is found in labiataeous and compositaeous plants as essential oils (de Lampasona et al., 1997;Tsuro et al., 2001; Gunawardena et al., 2002), and in leguminous and moraceous plants as diverse prenylated flavonoids (Barron and Ibrahim, 1996). Epstein and Poulter (1973) suggested very early that chrysanthemyl d...

Cloning and Characterization of Naringenin 8-Prenyltransferase, a Flavonoid-Specific Prenyltransferase of Sophora flavescens

“…Following transient expression, the fluorescence of SfN8DT1-GFP was localized to dotted organelles in both cell types, whose size and pattern were highly similar to that of isoprene synthase, a typical plastid protein, used as a positive control (Sasaki et al, 2005). These results suggested that SfN8DT-1 was localized to plastids as the native enzyme in S. flavescens (Zhao et al, 2003) as well as prenyltransferase of other plant species (Dhillon and Brown, 1976;Biggs et al, 1990;Fellermeier et al, 2001). …”

“…1). This intermediate, 8-dimethylallyl naringenin (8DN), is further hydroxylated to form leachianone G (LG) by 8DN 2#-hydroxylase (Yamamoto et al, 2001), and the second prenylation takes place at the prenyl side chain of LG catalyzed by LG 2$-dimethylallyltransferase (Zhao et al, 2003). Both prenylation reactions are Mg 21 dependent, plastid localized, and involve membranebound proteins.…”

“…The biosynthesis of SFG involves two prenylation reactions that have been biochemically determined to be associated with the crude membrane fraction of cultured cells (Yamamoto et al, 2000;Zhao et al, 2003). Naringenin is first prenylated at the 8-position with one dimethylallyl diphosphate (DMAPP; Fig.…”

Metabolism of administered (2RS)-naringenin in flavonoid-producing cultured cells of Sophora flavescens

“…On the other hand, in cells fed 0.3 mM (2RS)-naringenin notable accumulation of two intermediates 8PN and LG was observed, although the content of SFG was almost the same as that in cells fed 0.1 mM (2RS)-naringenin-fed cells. These results suggest that the second prenylation step catalyzed by leachianone G 2Љ-dimethylallyltransferase (Zhao et al 2003a) is one of the regulatory steps in SFG biosynthesis. The amount of TM, derived from 5-deoxyflavanone liquiritigenin but not from 5-hydroxylated naringenin, decreased by the addition of (2RS)-naringenin.…”

“…That is, naringenin was first dimethylallylated to afford 8-prenylnaringenin (8PN) by plastid-located naringenin 8-dimethylallyltransferase, a stereospecific enzyme for (Ϫ)-(2S)-naringenin (Yamamoto et al 2000), then hydroxylated to leachianone G (LG) by endoplasmic reticulum-associated cytochrome P450 mono-oxygenase, 8-prenylnaringenin 2Ј-hydroxylase (Yamamoto et al 2001a). Finally, another dimethylallyl moiety was transferred to the 2Љ position of LG by leachianone G 2Љ-dimethylallyltransferase exist in the plastid to form SFG (Zhao et al 2003a).…”